Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes. Graphical Abstract Highlights • Structure-based design of SARS-CoV-2 spike ectodomain and subdomain probes • On-column biotinylation and purification is enabled by affinity tag and HRV3C cleavage • Development of diverse molecular probes by “cut-and-paste” strategy • Application of probes to SARS-CoV-2 antibody discovery and immune evaluation SARS-CoV-2 spike probes are key to antibody discovery and vaccine evaluation. By using structure-based design, affinity purification, on-column biotinylation, and sequence-specific protease cleavage, Zhou et al. devise a strategy that allows for the rapid development of SARS-CoV-2 spike probes for B cell sorting, antibody discovery, and immune assessment.
【저자키워드】 COVID-19, Coronavirus disease 2019, antibody, structure-based design, biotinylated probe, human rhinovirus 3C, HRV3C protease, single-chain Fc,